Lipase-treated food products

ABSTRACT

Provided are a method for preparing a food product, which uses a lipase to catalyze the hydrolysis of fat and phospholipids in a base composition, and a food product produced by the method. The present method may increase the viscosity of the base composition having a low fat content and/or improve the stability of the food product even without added emulsifiers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication No. 63/070,011, filed on Aug. 25, 2020, the entire contentof which is hereby incorporated by reference.

BACKGROUND

Dairy products include suspensions of milk fat in an aqueous phase(including water and other milk components such as proteins, sugars,etc.), which may be converted into a blend from which other value-addedproducts such as sauces, soups, dressings, etc., are produced. Milkproducts may be homogenized to reduce the particle size of the milk fatand therefore increase the surface area over the liquid fraction,resulting in a more stable, viscous blend. However, these blends, evenwhen homogenized, tend to separate over the course of shelf life,resulting in fat rising to the top and heavier solids to the bottom of acontainer. In some cases, flocculation may occur whereby solids fall outof solution and precipitate to the bottom.

Dairy products have two major proteins: casein and whey. Either may beused for its nutritional value and/or its functionality. Casein isimportant for the body and flavor of dairy products. Various viscositiescan be achieved by adjusting the culturing process and selecting theparticular cultures to use in the fermentation process. Whey proteinsare heat coagulating proteins. Whey protein denatures under heatconditions resulting in changes in the body and the viscosity of thedairy product. Whey proteins are usually used as viscosity increasingingredients in soups, sauces, etc.

Dairy products are often formulated with other thickening agents such asgums and starches. Thickening agents, whether native or modified,contribute to mouthfeel and to the rheology of the finished product.Additionally, milk products are often formulated with emulsifying agentssuch as lecithin, mono and diglycerides, etc. to increase its stabilityand to prevent separation. Products which have been modified by theaddition of texturizing ingredients and emulsifiers may not be labelledas “clean.”

Lower fat dairy products (e.g., 10% or less milk fat content), whilehaving nutritional benefits compared to their higher fat counterparts,have inferior mouth feel, or lack the desired viscosity compared toproducts with regular or high fat content. Dairy products in general,regardless of fat content or processing method, lack enough stabilitywhich leads to separation or syneresis. The existing technology formodifying stability and viscosity often proves to be inadequate from aclean label perspective and/or unsuitable due to the addition ofnon-dairy ingredients or ingredients the market or industry will notpermit.

In view of the foregoing, there remains a need for food products (e.g.dairy products) having satisfactory stability, mouthfeel, and viscosity,as well as methods for producing such products. It is desired that suchmethods would not require added thickening or emulsifying agents,resulting in clean label and more cost-effective products.

SUMMARY

In an aspect, the present disclosure provides a method for preparing afood product, comprising mixing a lipase with a base compositioncomprising a phospholipid and 0% to about 99% by weight of fat, whereinthe lipase hydrolyzes the phospholipid, the milk fat, or a combinationthereof. In some embodiments, the base composition comprises a milk, amodified milk, a plant-based milk alternative, or a combination thereof.

In another aspect, provided is a food product produced by the method asdisclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a representative process flow chart.

FIG. 2 shows the viscosity values of 10% fat milk blends that wereuntreated (control) or treated with lipase.

FIGS. 3A-3G show the viscosity values of dairy products of various fatcontents (5%-35%), which were either not treated (Control) or treatedwith Enzyme 1 (Enzyme 1) or Enzyme 3 (Enzyme 3). Viscosity values (cP)were measured over time (Days) after inactivation of the enzymes atapproximately 40° F.

FIG. 4 shows representative results of a separation study of dairysamples at various fat contents (5%-35%) stored for 21 days. The sampleswere either not treated (Control) or treated with Enzyme 1 (Enzyme 1) orEnzyme 3 (Enzyme 3). The difference between the moisture level of thetop layer and the moisture level of the bottom layer was recorded(Moisture Difference (%)).

DETAILED DESCRIPTION

The present disclosure provides a method to modify the stability andviscosity of food products with a specific enzymatic treatment. Theherein described methods may eliminate the need for hydrocolloids andemulsifiers as additives in food products. For example, the viscosity ofa dairy blend having a low fat content (e.g., a 10% fat blend, such ashalf & half) may be modified to achieve a viscosity similar to that ofsour cream or even cream cheese. As a result, low fat blends may bemodified by the present method to have similar mouthfeel and viscosityas heavy cream and other high fat blends. In certain embodiments, theherein described methods do not involve culturing or denaturing anyproteins present naturally in milk, thereby allowing them to be intactfor further processing. The present technology may be applicable tovarious mixtures of milk and cream, and other combinations ofingredients that include or contain fat, which are standardized to awide range of fat level.

Before any embodiments of the invention are explained in detail, it isto be understood that the invention is not limited in its application tothe details of construction and the arrangement of components set forthin the following description. The invention is capable of otherembodiments and of being practiced or of being carried out in variousways.

1. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Suitable methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms “comprise(s),” “comprising,” “include(s),” “including,”“having,” “has,” “contain(s),” “containing,” and variants thereof, asused herein, are open-ended transitional phrases, terms, or words thatare meant to encompass the items listed thereafter and equivalentsthereof as well as additional items. The singular forms “a”, “and”, and“the” include plural references unless the context clearly dictatesotherwise. Where the term “comprising” is used, the present disclosurealso contemplates other embodiments “comprising,” “consisting of,” and“consisting essentially of,” the embodiments or elements presentedherein, whether explicitly set forth or not. Any numerical range recitedherein includes all values from the lower value to the upper value. Forexample, if a concentration range is stated as 1% to 50%, it is intendedthat values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., areexpressly enumerated in this specification. These are only examples ofwhat is specifically intended, and all possible combinations ofnumerical values between and including the lowest value and the highestvalue enumerated are to be considered to be expressly stated in thisapplication.

The modifier “about” used in connection with a quantity is inclusive ofthe stated value and has the meaning dictated by the context (forexample, it includes at least the degree of error associated with themeasurement of the particular quantity). The modifier “about” shouldalso be considered as disclosing the range defined by the absolutevalues of the two endpoints. For example, the expression “from about 2to about 4” also discloses the range “from 2 to 4.” The term “about” mayrefer to plus or minus 10% of the indicated number. For example, “about10%” may indicate a range of 9% to 11%, and “about 1” may mean from0.9-1.1. Other meanings of “about” may be apparent from the context,such as rounding off, so, for example “about 1” may also mean from 0.5to 1.4.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this disclosure, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito, 1999; Smith and March March's Advanced OrganicChemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock,Comprehensive Organic Transformations, VCH Publishers, Inc., New York,1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition,Cambridge University Press, Cambridge, 1987; the entire contents of eachof which are incorporated herein by reference.

The term “essentially free of” means that a composition contains acomponent in an amount of less than 1% by weight of the composition.This includes less than 0.5% by weight, less than 0.1% by weight, lessthan 0.05% by weight, or even less than 0.01% by weight. Compositions“essentially free of” a component also include a composition that iscompletely free of that component.

The term “lipase” as used herein refers to an enzyme which catalyzes thehydrolysis of ester bonds in lipid substrates such as fats, oils, andphospholipids. The lipase may include natural and recombinant enzymesknown in the art. The lipase may be obtained from animal tissues (suchas stomach or pancreas of cows, pigs, and goats), plants (such asvarious plant seeds), or microorganisms (such as bacteria, fungi, andyeast).

The term “lysophospholipid” as used herein means a derivative of aphospholipid after one or more of the fatty acid groups are removed.

The term “milk” as used herein refers to any animal milk (such as cowmilk) suitable for human consumption. The term “modified milk” as usedherein refers to any formulation derived from milk, in which the contentof at least one of the components of the milk, such as fat, protein,carbohydrates, ash, or water, is modified, or at least one ingredientnot present in the original milk is added, or both. For example, themodified milk may result from a milk after being treated by a known fatstandardization process. The milk or modified milk as used hereininclude any dry form (e.g., powder) of such milk or modified milk.

The term “plant-based milk alternative” refers to a product derived froma plant, for example by soaking, boiling, grinding, and/or blending thewhole plant or a part of the plant (e.g., a fruit and/or a seed), whichis suitable for human consumption. As a result, the plant-based milkalternative includes one or more nutrients, such proteins, fats,carbohydrates, fibers, vitamins, and minerals, etc., extracted from theplant. The plant-based milk alternative may have a texture or mouth feelsimilar to that of a milk. Examples of plant-based milk alternativesinclude oat milk, soy milk, coconut milk, almond milk, cashew milk, ricemilk, hemp milk, pea milk, hazelnut milk, and tiger nut milk, etc.,produced by manufacture processes known in the art. The plant-based milkalternatives as used herein include any dry form (e.g., powder) of suchproduct.

The term “phospholipid” as used herein includes organic molecules havingtwo fatty acid groups and a phosphate head group attached to a glycerolbackbone. The phosphate head group may include an organic moiety such ascholine, ethanolamine, inositol, or serine. The phospholipids describedherein include any naturally occurring phospholipids in cellularmembrane, such as phosphatidylcholine (PC), phosphatidylethanolamine(PE), phosphatidylinositol (PI), phosphatidylserine (PS), andsphingomyelin (SM).

2. Method

In one aspect, the present disclosure provides a method for preparing afood product. The method may comprise mixing a lipase with a basecomposition. The base composition may comprise a phospholipid and from0% to about 99% by weight of fat, wherein the lipase catalyzes ahydrolysis of the phospholipid, the fat, or a combination thereof.

The base composition may comprise a milk, a modified milk, a plant-basedmilk alternative, or a combination thereof. In some embodiments, thebase composition comprises a milk. In some embodiments, the basecomposition comprises a modified milk. In some embodiments, the basecomposition comprises a plant-based milk alternative.

The base composition may comprise phospholipids found in raw milk andother dairy products. In some embodiments, the base compositioncomprises a milk, a modified milk, a plant-based milk alternative, or acombination thereof, and the phospholipid of the base compositioncomprises the phospholipid present in the milk, the modified milk, aplant-based milk alternative, or the combination thereof. In someembodiments, the base composition comprises a milk, a modified milk, aplant-based milk alternative, or a combination thereof, and thephospholipid of the base composition consists the phospholipid presentin the milk, the modified milk, the plant-based milk alternative, or thecombination thereof.

The base composition may comprise about 0.01% to about 5% by weight ofphospholipid. In some embodiments, the base composition may include, byweight of the total phospholipid, about 8.0% to about 45.5%phosphatidylcholine, about 26.4% to about 72.3%phosphatidylethanolamine, about 1.4% to about 14.1%phosphatidylinositol, about 2.0% to about 16.1% phosphatidylserine, andabout 4.1% to about 29.2% sphingomyelin.

The base composition may include natural milk fat (or butter fat) or fatof other sources, which includes triglycerides with various fatty acidcomponents. In some embodiments, the base composition comprises a milk,a modified milk, a plant-based milk alternative, or a combinationthereof, and the fat of the base composition comprises the fat presentin the milk, the modified milk, a plant-based milk alternative, or thecombination thereof. In some embodiments, the base composition comprisesa milk, a modified milk, a plant-based milk alternative, or acombination thereof, and the fat of the base composition consists thefat present in the milk, the modified milk, the plant-based milkalternative, or the combination thereof.

The base composition may comprise about 0.1% to about 99% by weight offat, including about 0.1% to about 80%, about 0.1% to about 60%, about0.1% to about 45%, about 1% to about 45%, about 1% to about 40%, about5% to about 40%, about 5% to about 30%, about 5% to about 25%, or about5% to about 15% by weight of fat. The base composition may compriseabout 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, or about 40% by weight of fat. In certain embodiments, the basecomposition comprises about 10% by weight of fat.

In certain embodiments, the base composition comprises a modified milk,the phospholipid of the base composition consists of the phospholipidpresent in the modified milk, and the fat of the base compositionconsists of the fat present in the modified milk. In such embodiments,the fat content of the base composition may be about 0.1% to about 60%by weight, such as about 0.1% to about 45%, about 1% to about 45%, about1% to about 40%, about 5% to about 40%, about 5% to about 30%, about 5%to about 25%, or about 5% to about 15% by weight. The modified milk maybe derived from a raw milk using known techniques. For example, themodified milk may be produced from a raw milk following a fatstandardization process, in which the fat content is adjusted to apre-determined level.

The lipase as disclosed herein includes, but are not limited to,triacylglycerol lipases capable of hydrolyzing triglycerides andphospholipases capable of hydrolyzing phospholipids. The triacylglycerollipase may include, for example, enzymes under Enzyme Commission numberEC 3.1.1.3 or E.C. 232-619-9, enzymes under Chemical Abstracts ServiceRegistry Number (CAS No.) 9001-62-1, and enzymes under InternationalUnion of Biochemistry number (IUB No.) 3.1.1.3. The phospholipase maybe, for example, phospholipase A1, phospholipase A2, phospholipase B,phospholipase C, and/or phospholipase D. The lipase may contain bothtriacylglycerol lipase and phospholipase properties. The lipase maycatalyze the hydrolysis of one or two fatty acyl chains from the fat orthe phospholipid, the hydrolysis of the phosphate head group from thephospholipid, or a combination thereof. In some embodiments, the lipaseis a phospholipase that catalyzes the hydrolysis of one fatty acyl chainfrom the phospholipid to produce a lysophospholipid.

In some embodiments, one lipase (a triacylglycerol lipases or aphospholipase) is used to catalyze the hydrolysis of the fat and/or thephospholipid. In some embodiments, two or more lipases are used tocatalyze the hydrolysis of the fat and/or the phospholipid. In someembodiments, at least a portion of the phospholipids in the basecomposition is hydrolyzed. In some embodiments, at least a portion ofthe fat in the base composition is hydrolyzed. In some embodiments, atleast a portion of the phospholipids and at least a portion of the fatin the base composition are hydrolyzed.

Suitable lipases include those obtained from animal, plant, bacteria,fungus, or genetically engineered microorganisms. In some embodiments,the lipase includes one or more phospholipases and/or one or moretriacylglycerol lipases, each of which is obtained from an animal, aplant, or microbial fermentation.

The base composition may be pasteurized and homogenized using knowntechniques before mixing with the lipase. The mixing may be carried outusing known techniques. Other procedures may be utilized to facilitatethe mixing of the base composition and the lipase, as well as thehydrolysis of fat and/or phospholipids catalyzed by the lipase. Suchprocedures may include, for example, heating, cooling, stirring,shaking, incubating, pH adjustment, and other known techniques.

The lipase-catalyzed hydrolysis may be carried out at a temperature ofabout 30° F. to about 160° F., such as about 50° F. to about 150° F., orabout 75° F. to about 125° F. The hydrolysis may be carried out at about35° F., about 40° F., about 45° F., about 50° F., about 55° F., about60° F., about 65° F., about 70° F., about 75° F., about 80° F., about85° F., about 90° F., about 95° F., about 100° F., about 105° F., about110° F., about 115° F., about 120° F., about 125° F., about 130° F.,about 135° F., about 140° F., or about 145° F.

The lipase-catalyzed hydrolysis may be carried out for a time period ofabout 10 minutes to about 150 minutes. The hydrolysis may be carried outfor a time period of about 30 minutes, about 40 minutes, about 50minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90minutes, about 100 minutes, about 110 minutes, about 120 minutes, about130 minutes, or about 140 minutes. In some embodiments, the hydrolysiswas carried out at a temperature of about 75° F. to about 125° F. for aperiod of about 30 minutes.

The present method may further include adding a flavorant, such as thenatural or artificial flavor compounds known in the art. The flavorantmay improve the sensory quality (e.g., pleasant taste and smell) of thefinal product. Suitable flavorants include natural and artificialflavors, such as salt (NaCl), sugar, herb extracts, fruit extracts,vegetable extracts, spices, or a combination thereof. The flavorant maybe added before or after the lipase-catalyzed hydrolysis of the basecomposition. In some embodiments, the flavorant is added after thelipase-catalyzed hydrolysis.

The present method may further include adding one or more antioxidantsto control the oxidation level during production or storage of theproduct. Suitable antioxidants include compounds such as phenols,carotenoids, curcumins, xanthones, vitamins A, E and C, citric acid,flavonoids, terpenoids, lignans, sulfides, plant sterols, andcombinations thereof. The one or more antioxidants may be added beforeor after the lipase-catalyzed hydrolysis of the base composition. Insome embodiments, the antioxidant is added to the base compositionbefore the lipase-catalyzed hydrolysis.

The present method may further include a step of inactivating the lipasefollowing the hydrolysis. The inactivation may be performed using knowntechniques, such as heat treatment. In some embodiments, the lipase isinactivated by heating at a temperature of at least 160° F., including,for example, at least 165° F., at least 170° F., at least 180° F., or atleast 200° F. In some embodiments, the lipase is inactivated by heatingat temperature of about 165° F.

Viscosity is a measurement of a fluid's resistance to flow. Viscosityvalues may be used describe the mouth feel of a food product. In dairyproducts, viscosity at a given temperature is dependent upon itscomposition and the physical state of its colloidally dispersedsubstances, including fat. Homogenization may increase a dairy product'sviscosity due to the increased surface area and dispersed state of thefat. Advantageously, the present method may significantly increase theviscosity of a base composition having a lower fat content (e.g., 10%fat) to a level similar to that of products having much higher fatcontent, such as sour cream (about 20% fat) or even cream cheese (about35% fat). As a result, low fat base compositions (such as low fat milk)may be modified by the present method to have similar mouthfeel andviscosity comparable to those of heavy cream and high fat products.

In addition, the present method may improve the stability of the foodproduct, with or without using hydrocolloids and emulsifiers asadditives. For example, phase separation occurs in dairy products overthe course of shelf life because cream, or butterfat, is lighter thanother components in milk. Homogenization may reduce phase separation byreducing the size of fat globules, thus increasing their surface areaand resulting in a more uniform and stable distribution of fatthroughout the aqueous phase. Emulsifiers may be utilized to reduce thedegree of separation by decreasing the surface tension between the waterand oil phases. Emulsifiers function by having both hydrophilic andhydrophobic properties to interact with both phases (also known asamphipathic character).

Without being limited by any theory, the lysophospholipids produced fromphospholipids (e.g., catalyzed by the phospholipase) may function asendogenous emulsifiers that lead to increased stability of the foodproduct, such as a dairy product. In addition, the production ofmonoglycerides, diglycerides, and free fatty acids from triglycerides(e.g., catalyzed by the triacylglycerol lipase) may increase the surfacearea of the fat, which leads to increased body and further improves thestability of the dairy product. Most of the phospholipids in milk arelocated in the milk fat globule membrane (MFGM) or the outer layer ofmilk fat that encapsulates micelles. The lipase may cause disintegrationof these membranes by hydrolyzing the membrane phospholipids, therebyreleasing the encapsulated amphipathic fat molecules. Thus, the methoddescribed herein may reduce phase separation and improve the stabilityof the food product, without the use of added emulsifiers.

The present method may be performed under mild conditions so as to keepthe proteins in the food product (such as milk proteins) intact,allowing the food product to maintain its protein composition, which maybe further processed or incorporated into other products.

3. Composition

In another aspect, the present disclosure provides a food productproduced by the method as described herein. In some embodiments, thefood product is a dairy product (such as a dairy blend) produced frommilk.

The food product may have a fat content of about 1% to about 90% byweight. The food product may have a fat content of at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least85% by weight. The food product may have a fat content of at most 85%,at most 80%, at most 75%, at most 70%, at most 65%, at most 60%, at most55%, at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, atmost 25%, at most 20%, at most 15%, at most 10%, or at most 5% byweight. The food product may have a fat content of about 1% to about80%, about 1% to about 60%, about 1% to about 45%, about 2% to about45%, about 5% to about 45%, about 5% to about 40%, about 5% to about35%, about 5% to about 30%, about 5% to about 25%, about 5% to about20%, or about 5% to about 15% by weight. In some embodiments, the foodproduct has a fat content of about 5% to about 25% by weight. In someembodiments, the food product has a fat content of about 2%, about 5%,about 10%, about 15%, about 20%, about 25%, or about 30% by weight.

The food product may have a viscosity of about 1000 cP to about 1500000cP at 40° F. The food product may have a viscosity of at least 1000 cP,at least 5000 cP, at least 10000 cP, at least 50000 cP, at least 100000cP, at least 200000 cP, at least 300000 cP, at least 400000 cP, at least500000 cP, at least 600000 cP, at least 700000 cP, at least 800000 cP,at least 1000000 cP, or at least 1200000 cP at 40° F. The food productmay have a viscosity of about 10000 cP to about 1200000 cP, about 20000cP to about 1200000 cP, about 20000 cP to about 1000000 cP, about 20000cP to about 800000 cP, about 20000 cP to about 600000 cP, about 20000 cPto about 400000 cP, about 20000 cP to about 200000 cP, or about 20000 cPto about 100000 cP at 40° F. The food product may have a viscosity ofabout 100000 cP to about 1000000 cP, about 100000 cP to about 800000 cP,about 100000 cP to about 600000 cP, about 100000 cP to about 500000 cP,about 100000 cP to about 400000 cP, about 100000 cP to about 300000 cP,or about 100000 cP to about 200000 cP at 40° F.

The food product may be stable (e.g., with minimal or no phaseseparation) for at least 10 days in a closed container at temperaturebetween 0° C. to about 25° C. The food product may be stored underrefrigerated conditions (e.g., about 4° C. or 40° F.). The stability ofthe food product may include physical stability (e.g., as demonstratedby minimal or no phase separation) and chemical stability (e.g., asdemonstrated by low microbial growth) during storage. In someembodiments, the food product may be stable for at least 15 days, atleast 20 days, at least 30 days, at least 40 days, at least 50 days, oreven at least 60 days at 40° F.

The food product produced by the method as described herein may beessentially free of emulsifiers or texturizing agents that are added toknown products. For example, the food product may be essentially free ofadded emulsifiers, such as soy and egg lecithin, mono- and diglycerides,polysorbates, carrageenan, guar gum, or canola oil. The food product maybe essentially free of added texturing ingredients, such as starches,soy proteins, alginates, pectin, xanthan gum, carrageenan, andgalactomannans. In some embodiments, the food product does not includeany added emulsifiers or texturizing ingredients.

The present food product, as a result of the lipase treatment describedherein, may enhance salt perception compared to a product that is nottreated by lipase. The enhancement of salt perception may be measured bya panel that is trained using standards with known concentrations ofsalt. The panel reports a perceived salt concentration for a sample,which indicates the observed salt perception in that sample. In someembodiments, the panel indicates at least 1.5-fold, at least 5-fold, oreven at least 10-fold increase in salt perception for the lipase treatedsamples, compared to the control sample containing the same amount ofsalt.

The food product may further comprise other food additives known in theart, such as antioxidants, flavorants, vitamins, colorants, sweeteners,preservatives, or a combination thereof.

The food product produced by the method as described herein may be adairy blend. The food product may be in the form of a liquid, a cream, asemi-solid, or a solid. The food product may be included to formulate aready-to-use food composition for cooking or consumption, such as asauce, a salad dressing, a drink, or a soup. The herein described foodproduct (e.g., 10% fat), as a result of lipase treatment describedherein, may achieve sensory performance (such as flavor, creaminess, andoverall liking) that is comparable to a control product having a muchhigher fat level (e.g., 40% fat).

EXAMPLES Example 1. Lipase Treatment of a Milk Composition

All industrial and lab instruments for dairy product processing as usedherein are commercially available, including separator, blend tank,scale, pasteurizer, homogenizer, cooling press, heating tank, steamjacketed kettle, tote, and metalized dairy bags.

The lipase enzymes tested are shown below.

Enzyme 1 Enzyme 3 Enzyme 4 Enzyme 5 Enzyme 6 Protein E.C. 3.1.1.3 CASNo. 9001- CAS No. 9001- CAS No. 9001- CAS No. 9001- ID 62-1 62-1 62-162-1 IUB No. 3.1.1.3 E.C. 232-619-9 Activity PhospholipaseTriacylglycerol Triacylglycerol Triacylglycerol Triacylglycerol type A1lipase lipase lipase lipase Origin Microbial Microbial Animal MicrobialAnimal Fermentation Fermentation Fermentation

A representative procedure is shown in FIG. 1. Cream and skim milk wereseparated using a separator. Subsequently, a blend having a 10% milk fatwas obtained following a fat standardization step. Antioxidant (0.2% wasadded), and the resulting composition was pasteurized at 165° F. (with a40-second hold). The pasteurized composition was subjected to ahomogenization process (1500 PSI first stage, 500 PSI second stage), andthe homogenized composition was incubated with the lipase for 30 minutesat 75-125° F. (Enzyme 1: 0.2-0.54% by weight; Enzyme 3: 0.2-0.45% byweight). The enzyme was inactivated by heating at 165° F. (with a6-minute hold), and the resulting composition was then cooled to below40° F., packed, and was allowed to age and stored in a manner to controlexposure to light.

The viscosity values of the untreated (control) and the enzyme treated10% fat milk blends were measured over time (FIG. 2). The results showedthat the change in viscosity was solely related to the enzymatictreatment and that other factors (such as protein denaturation) was notthe cause of such change. Both untreated and treated blends weresubjected to the same time and temperature variables.

Example 2. Viscosity Study

The viscosity values of the products from the lipase treatment processwere studied. A rheometer was used to measure the way in which a liquid,suspension or slurry flows in response to applied forces. It is used forthose fluids which cannot be defined by a single value of viscosity andtherefore requires more parameters to be set and measured than is thecase for a viscometer. In the absence of a rheometer, a viscometer wasused to measure fluid viscosity in centipoise (cP) at given shear rates.

The process started with raw milk that is separated into cream and skimfractions. The resulting intermediate was standardized to a certain fatcontent (5%-35%) and controlled for light. The fluid dairy was sentthrough a high-temperature short-time (HTST) process where it ispasteurized in adherence to Pasteurized Milk Ordinance (PMO) standardsand homogenized. The fluid dairy was either not treated (Control),treated with Enzyme 1 (Enzyme 1), or treated with Enzyme 3 (Enzyme 3) ata specific time and temperature as described above. The product was thensubjected to a second heat treatment process for inline inactivation,after which the product was cooled. The cooled product was thenpackaged, aged, and stored.

The viscosities for the products from each fat content under eachtreatment (Control, Enzyme 1, Enzyme 3) were measured over time (a totalof 21 trials). Typically, after enzyme inactivation, the products wereseparately agitated and poured into 15 oz jars and labeled accordingly.Samples were stored in a cooler at 40° F. At different time points (1,3, 5, 7, 14, and 21 days), viscosity for each product was measured usinga Brookfield viscometer. Different RV Spindles and T-Bar Spindles wereused depending on the viscosity of the product. As shown in FIGS. 3A-3G,the lipase treatment significantly increases the viscosity of dairyproducts (higher cP values), allowing them to emulate the viscosity andmouth feel of higher fat products.

Example 3. Separation Study

Further tests were conducted to measure the degree of separation in thelipase treated products as described above. Typically, products fromeach fat content (5%-35%) under each treatment (Control, Enzyme 1,Enzyme 3) were separately agitated and poured into 100 mL graduatedcylinders and labeled accordingly. Samples were stored in a cooler at40° F. After 21 days, a picture was taken for each sample to visuallymeasure the separation of the sample into a top layer and a bottomlayer. A glass pipette fitted with a rubber bulb was used to take analiquot from each of the top and bottom layers. Moisture contents(percentage) in the aliquots were measured using a moisture analyzer.The difference between the moisture level of the top layer and themoisture level of the bottom layer was recorded (Moisture Difference(%)), as shown in FIG. 4. Since fat is lighter than other components inmilk, a greater moisture difference between the top layer and the bottomlayer indicates greater separation.

Example 4. Lysophospholipid Production

The lipase treatment produced lysophospholipids from the naturallyoccurring fat matrix available in dairy products. It is hypothesizedthat the lysophospholipids produced from phospholipids function asendogenous emulsifiers and lead to increased stability of the dairyproduct. In a representative study, raw milk was separated into creamand skim fractions, and was then standardized to 10% fat content andcontrolled for light. The fluid dairy was sent through ahigh-temperature short-time (HTST) process where it is pasteurized inadherence to Pasteurized Milk Ordinance (PMO) standards and homogenized.The fluid dairy was then either not treated (Control) or treated withEnzyme 1 at a specific time and temperature as described above. Theproduct was then subjected to a second heat treatment process for inlineinactivation, after which the product was cooled. The cooled product wasthen packaged, aged, and stored.

Production of lysophospholipids was measured by quantitative 31P-NMRspectroscopy using a Bruker Avance III HD 600 MHz NMR spectrometer withautomated sample changer and BBO cryoprobe and an internal standard oftriphenyl phosphate (TPP). As shown in Table 1, the lipase treatmentresulted in significant reduction of phosphatidylcholine andphosphatidylethanolamine contents and production of2-lysophosphatidylcholine as a major lysophospholipid. The nondetectableamount of lysophosphatidylethanolamine may indicate a completeconsumption of phosphatidylethanolamine by the lipase. The relativelyconstant amount of sphingomyelin in both Control and Enzyme 1 samplesmay indicate that the sphingomyelin was not significantly metabolized bythe enzyme.

TABLE 1 Molecular Control Enzyme 1 Phospholipid Weight (mol. %) (mol. %)Phosphatidylcholine 757.0 33.9 6.1 2-Lysophosphatidylcholine 515.0 n.d.10.3 Phosphatidylethanolamine 755.0 6.8 n.d.Lysophosphatidylethanolamine 470.0 n.d. n.d. Sphingomyelin 770.0 27.641.9

Example 5. Shelf Life Study

According to the Pasteurized Milk Ordinance (PMO), the terms“pasteurization”, “pasteurized” and similar terms shall mean the processof heating every particle of milk or milk product, in properly designedand operated equipment, to one (1) of the temperatures given in thefollowing chart and held continuously at or above that temperature forat least the corresponding specified time.

Batch (Vat) Pasteurization Temperature Time 63° C. (145° F.) 30 minutesContinuous Flow (HTST and HHST) Pasteurization Temperature Time 72° C.(161° F.)  15 seconds 89° C. (191° F.) 1.0 seconds 90° C. (194° F.) 0.5seconds 94° C. (201° F.) 0.1 seconds 96° C. (204° F.) 0.05 seconds  100°C. (212° F.)  0.01 seconds 

If the fat content of the milk product is 10% or greater, or a totalsolids of 18% or greater, or if it contains added sweeteners, thespecified temperature shall be increased by 3° C. (5° F.). Under themost favorable processing and storage conditions, this HTSTpasteurization process is typically capable of extending the shelf lifeof fresh milk products for up to 3 weeks (21 days) depending on theinitial microbiological quality of the raw milk, as demonstrated byfresh milk products on the shelf. Other methods are available forextending the shelf life of milk, but they typically involve changingthe heating regime. The other traditionally applied process is ultrahigh temperature (UHT), which used a high temperature (>135° C.) for 1-2seconds. UHT products can be stored at ambient temperatures and whileboasting a longer shelf life, the increased temperature in the UHTprocess leads to organoleptic changes in the product like a “cooked”flavor note.

The shelf stability of the dairy produced by the present method wasexamined. The process started with raw milk that is separated into creamand skim fractions. It is standardized to a 10% fat content andcontrolled for light. The fluid dairy is sent through a high-temperatureshort-time (HTST) process where it is pasteurized in adherence toPasteurized Milk Ordinance (PMO) standards and homogenized. The fluiddairy was either not treated (Control) or treated with a lipase (Enzyme5 or Enzyme 6) (Treated) at a specific time and temperature as describedabove. The product was then subjected to a second heat treatment processfor inline inactivation and cooling. The cooled product was packaged,aged, and stored.

In order to accurately measure an extension in shelf life, each samplewas individually packed in twenty-four (24) separate sterile samplecontainers. Samples were stored in a cooler at 40° F. At different timepoints (0, 7, 14, 16, 19, 21, 23, 26, 27, 29, 30, 33, 35, 37, 40, 42,44, 47, 49, 51, 54, 56, 58, and 61 days), the containers were taken outof the cooler and were sent to a lab for microbiological testingaccording to Association of Official Analytical Chemists (AOAC) methodsand include aerobic plate count (APC) according to AOAC 990.12, totalcoliforms (C) according to AOAC 991.14, and yeast (Y) and mold (M)according to AOAC 2014.05 (Table 2). For reference, the PMO declares thebacterial limit is 20,000 colony forming units (CFU)/g for pasteurizedmilk and/or milk products to be considered Grade “A”.

As shown by the APC measurement, the Control sample confirmed a typicalshelf life of 21 days for dairy products (APC<1,000,000). After 21 days,there was a steady microbiological growth in the Control sample (APCcount increase to >40,000,000). By day 40, the Control sample wascoagulated due to significant microbiological growth. In contrast, theenzyme treated product of the present method remained stable andmaintained low APC count (<20,000) for at least 60 days. These resultsdemonstrate that enzyme treatment process may effectively control themicrobiological counts in the matrix, thus extending the shelf life offresh dairy products. Preventing microbial growth and extending theshelf life can alleviate costs related to spoilage.

TABLE 2 Day Enzyme 5 Enzyme 6 Control (age) C APC Y M C APC Y M C APC YM 0 <1 3 <10 <10 <1 350 <10 <10 <1 0 7 <10 10 <10 <10 <10 5,800 <10 <10<10 <10 <10 <10 14 <10 <10 <10 <10 <10 1,700 <10 <10 <10 710 <10 <10 16<10 <10 <10 <10 <10 1,600 <10 <10 <10 160,000,000 * <10 <10 19 <10 <10<10 <10 <10 1,400 <10 <10 <10 44,000 <10 <10 21 <10 <10 <10 <10 <101,100 <10 <10 18,000 640,000,000 * <10 <10 23 <10 <10 <10 <10 <10 890<10 <10 <10 1,100,000 <10 <10 26 <10 10 <10 <10 <10 620 <10 <10<10 >2,500,000,000 * <10 <10 27 <10 10 <10 <10 <10 120,000,000 * <10 <10<10 13,000,000 <10 <10 29 <10 150 <10 <10 <10 470 <10 <10 <10 43,000,000<10 <10 30 <10 <10 <10 <10 <10 620 <10 <10 <10 31,000,000 <10 <10 33 <10<10 <10 <10 <10 390 <10 <10 <10 37,000,000 <10 <10 35 <10 <10 <10 <10<10 300 <10 <10 <10 75,000,000 <10 <10 37 <10 20 <10 <10 <10 16,000 <10<10 <10 43,000,000 <10 <10 40 <10 <10 <10 <10 <10 220 <10 <10 <108,100,000 <10 <10 42 <10 10 <10 <10 <10 300 <10 <10 44 <10 <10 <10 <10<10 5,000 <10 <10 47 <10 10 <10 <10 <10 4,700 <10 <10 49 <10 20 <10 <10<10 14,000 <10 <10 51 <10 <10 <10 <10 <10 3,300 <10 <10 54 <10 10 <10<10 <10 7,900 <10 <10 56 <10 2,700 <10 <10 <10 <10 <10 <10 58 <10 10 <10<10 <10 12,000 <10 <10 61 <10 <10 <10 <10 <10 33,000 <10 <10 * numberswhich may reflect accidental contamination during preparation and/orsampling. Example 6. Enhancement of Salt Perception Study

Each of the four basic tastes (sweet, salt, sour, and bitter) can beinfluenced by the entire food matrix, not just a single ingredient. Forexample, sweetness perception is not only increased by increasing sugarcontent but also by pairing it with sour thus increasing the overallintensity of both. Here, the capacity of the present dairy product toenhance salt perception was examined.

In this study, the process started with raw milk that is separated intocream and skim fractions. The intermediate product was then standardizedto a 10% fat content and controlled for light. The fluid dairy was sentthrough a high-temperature short-time (HTST) process where it ispasteurized in adherence to Pasteurized Milk Ordinance (PMO) standardsand homogenized. The fluid dairy was either not treated (Control) ortreated with one version of a lipase (Enzyme 6) at a specific time andtemperature as described above. The product was then subjected to asecond heat treatment process for inline inactivation and cooling. Thecooled product was then packaged, aged, and stored.

In order to accurately measure salt perception, a trained panel wassetup. Panelists were chosen based on their ability to pass a triangletest, which rates their ability to distinguish between three samples. Atriangle test is a discriminative method where a panelist is presentedwith one different and two alike samples. Samples were presented in allpossible permutations to reduce any presentation order biases andpanelists were instructed to taste the samples from left to right tokeep consistencies. The triangle test samples consisted of two dairyblends, one with 1.15% salt added and another with 1.39% salt added.These numbers were chosen because the difference threshold for humans istypically 15%, which is the extent of change in the stimulus necessaryto produce a reliably noticeable difference. That percentage wasincreased to allow for more panelists in the trained panel. Seven (7)panelists passed the triangle test and were admitted in the trainedpanel.

Training was divided into nine (9) separate training sessions, with eachsession lasting roughly 30 minutes. Panelists were trained using five(5) separate reference standards, which consisted of dairy blends with aset amount of salt. The dairy blend reference standards contained 0.25%,0.75%, 1.00%, 1.25%, and 1.75% salt respectively. The goal of thetrained panel is to have participants familiarize themselves with thereference standards and make sure that panelists can accurately rate aproduct on the scale. The first training session was introducing thestandards. The second training session determined if the panelists couldplace the reference standards in order of increasing intensity. Thefollowing four (4) training sessions, panelists were given three (3)samples each and were asked to place them on the scale with referencestandards available. The final three (3) training sessions, panelistswere given three (3) samples each and were asked to place them on thescale without reference standards available. At this point, the trainingwas complete and panelists were considered trained once they were ableto accurately place samples on the scale provided.

The analysis was conducted by presenting the control sample (Control)and enzyme treated samples (Treated) together with a set amount of saltadded in two separate tests, with at least 10 minutes in between toallow for palette cleansing. Panelists were instructed to rate the twosamples on a scale without reference standards available. There was asignificant difference in salt intensity perception for the Control andTreated samples at alpha=0.05 for 0% salt and 0.50% salt addition (Table3). These results indicate that the dairy product produced by thepresent method may enhance salt perception, thereby reducing the sodiumcontent in the final product necessary to achieve the same overallflavor.

TABLE 3 Control Treated Control Treated Salt % 0 0 0.5 0.5 Mean 0.0360.21 0.357 0.657 Variance 0.004 0.002 0.025 0.048 p-value (one-tail ttest) 0.0003 0.0002

Example 7. Product Performance Study

Performance of a representative cream produced by the present technology(10% fat, using Enzyme 4) was compared to a control cream (40% fat) inan Alfredo pasta sauce product. The ingredients of the control sauce(Control) and the sauce containing a cream produced by the above method(Treated) are as follows.

Ingredient Control (wt %) Treated (wt %) Skim Milk 44.56 44.56 Cream(40% Fat) 41.10 — Cream (10% fat), treated — 41.10 Parmesan Cheese 11.2711.27 Xanthan Gum 0.09 0.09 Garlic Powder 0.27 0.27 Black Pepper 0.140.14 Food Starch - Modified 1.77 1.77 Salt 0.81 0.81 Total 100.00 100.00Total fat 16.47 4.14

A sensory panel was implemented for an accurate, non-bias, andcontrolled analysis of the two products. The sensory panel used a9-point hedonic scale (shown below) which is the most widely used scalefor testing and measuring consumer acceptability of food.

9-Point Hedonic Scale 9 Like Extremely 8 Like very Much 7 LikeModerately 6 Like Slightly 5 Neither Like nor Dislike 4 Dislike Slightly3 Dislike Moderately 2 Dislike Very Much 1 Dislike Extremely

Each sample was coded with a randomly generated 3-digit number, so thepanelists were not biased and the tasting was considered blind. Sampleswere presented in all possible permutations to reduce any presentationorder biases and panelists were instructed to taste the samples fromleft to right to keep consistencies. Panelists were screened andselected based on being a regular consumer, meaning they consumedalfredo at least once a month. With this selection criteria, ninety-nine(99) panelists were selected to participate. Panelists were asked toscore the two Alfredo pasta sauces on four different sensory categories:overall liking, overall flavor liking, sauce overall liking, andcreaminess liking. While “overall liking” and “overall flavor liking”are categories that are commonly used in a hedonic sensory test, “sauceoverall liking” and “creaminess liking” were added as an emphasis onattributes related to Alfredo pasta sauces. With a score of six (6) as“Like Slightly” and a score of seven (7) as “Like Moderately”, theaverage results of the panelists' responses are showed in Table 4.

TABLE 4 Category Control Treated Overall Liking 7.26 6.90 Overall FlavorLiking 7.07 6.80 Sauce Overall Liking 7.12 6.77 Creaminess Liking 6.976.56

These results show that the Alfredo pasta sauces containing the controlcream (40% fat) and the cream produced by the present method (10% fat)demonstrated relatively equal sensory performance. Despite theirdifference in “Creaminess Liking” (a difference of 0.41 in theirscores), both products were rated close to “Like Moderately.”

For reasons of completeness, various aspects of the disclosure are setout in the following numbered clauses:

Clause 1. A method for preparing a food product, comprising mixing alipase with a base composition comprising a phospholipid and 0% to about99% by weight of fat, wherein the lipase catalyzes a hydrolysis of thephospholipid, the fat, or a combination thereof.

Clause 2. The method of clause 1, wherein the base composition comprisesa milk, a modified milk, a plant-based milk alternative, or acombination thereof.

Clause 3. The method of any one of clauses 1-2, wherein the basecomposition comprises a modified milk.

Clause 4. The method of any one of clauses 1-3, wherein the basecomposition comprises 0.1% to about 45% by weight of fat.

Clause 5. The method of any one of clauses 1-4, further comprisinginactivating the lipase after the hydrolysis.

Clause 6. The method of any one of clauses 1-5, wherein the hydrolysisis carried out at a temperature of about 50° F. to about 150° F.

Clause 7. The method of any one of clauses 1-6, wherein the hydrolysisis carried out for a time period of about 10 minutes to about 150minutes.

Clause 8. The method of any one of clauses 1-7, wherein the phospholipidis hydrolyzed to produce a lysophospholipid.

Clause 9. The method of any one of clauses 1-8, further comprisingadding a flavorant.

Clause 10. The method of any one of clauses 1-9, further comprisingadding an antioxidant.

Clause 11. A food product produced by the method of any one of clauses1-10.

Clause 12. The food product of clause 11, which is essentially free ofadded emulsifiers or texturizing agents.

Clause 13. The food product of any one of clauses 11-12, comprisingabout 5% to about 25% by weight of fat.

Clause 14. The food product of any one of clauses 11-13, having aviscosity of about 1000 cP to about 1200000 cP at 40° F.

Clause 15. The food product of any one of clauses 11-14, having aviscosity of about 20000 cP to about 600000 cP at 40° F.

Clause 16. The food product of any one of clauses 11-15, which is stablefor at least 30 days at 40° F.

Clause 17. The food product of any one of clauses 11-15, which is in theform of a cream.

Clause 18. The food product of clause 17, comprising about 10% by weightof fat.

Clause 19. A food composition comprising the food product of clause 11.

Clause 20. The food composition of clause 19, which is a sauce, a saladdressing, a drink, or a soup.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe following claims.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A method for preparing a food product, comprisingmixing a lipase with a base composition comprising a phospholipid and 0%to about 99% by weight of fat, wherein the lipase catalyzes a hydrolysisof the phospholipid, the fat, or a combination thereof.
 2. The method ofclaim 1, wherein the base composition comprises a milk, a modified milk,a plant-based milk alternative, or a combination thereof.
 3. The methodof claim 1, wherein the base composition comprises a modified milk. 4.The method of claim 1, wherein the base composition comprises 0.1% toabout 45% by weight of fat.
 5. The method of claim 1, further comprisinginactivating the lipase after the hydrolysis.
 6. The method of claim 1,wherein the hydrolysis is carried out at a temperature of about 50° F.to about 150° F.
 7. The method of claim 1, wherein the hydrolysis iscarried out for a time period of about 10 minutes to about 150 minutes.8. The method of claim 1, wherein the phospholipid is hydrolyzed toproduce a lysophospholipid.
 9. The method of claim 1, further comprisingadding a flavorant.
 10. The method of claim 1, further comprising addingan antioxidant.
 11. A food product produced by the method of claim 1.12. The food product of claim 11, which is essentially free of addedemulsifiers or texturizing agents.
 13. The food product of claim 11,comprising about 5% to about 25% by weight of fat.
 14. The food productof claim 11, having a viscosity of about 1000 cP to about 1200000 cP at40° F.
 15. The food product of claim 11, having a viscosity of about20000 cP to about 600000 cP at 40° F.
 16. The food product of claim 11,which is stable for at least 30 days at 40° F.
 17. The food product ofclaim 11, which is in the form of a cream.
 18. The food product of claim17, comprising about 10% by weight of fat.
 19. A food compositioncomprising the food product of claim
 11. 20. The food composition ofclaim 19, which is a sauce, a salad dressing, a drink, or a soup.